本文選題：DLX1 + 骨肉瘤�。� 參考：《重慶醫(yī)科大學(xué)》2017年碩士論文

【摘要】：第一部分DLX1過(guò)表達(dá)對(duì)人骨肉瘤細(xì)胞惡性生物學(xué)行為及成骨分化的影響目的探討DLX1過(guò)表達(dá)對(duì)人骨肉瘤細(xì)胞MG63惡性生物學(xué)行為及成骨分化的影響。方法采用構(gòu)建有DLX1基因的重組腺病毒(adenovirus,Ad)Ad DLX1和空載腺病毒Ad RFP,分別感染人骨肉瘤細(xì)胞MG63,分為DLX1實(shí)驗(yàn)組(Ad DLX1感染組)和RFP對(duì)照組(Ad RFP感染組),觀察細(xì)胞熒光表達(dá)情況并以RT-PCR和Western blot驗(yàn)證DLX1的表達(dá)情況;Transwell實(shí)驗(yàn)檢測(cè)MG63細(xì)胞遷移和侵襲;DAPI染色和流式細(xì)胞術(shù)檢測(cè)MG63細(xì)胞凋亡;CCK-8檢測(cè)MG63細(xì)胞增殖;堿性磷酸酶(alkaline phosphatase,ALP)染色和ALP讀數(shù)分析MG63細(xì)胞早期成骨能力,Western blot檢測(cè)骨橋蛋白(osteopontin,OPN)的蛋白表達(dá),茜素紅染色檢測(cè)細(xì)胞晚期成骨能力;RT-PCR初篩相關(guān)信號(hào)通路,Western blot驗(yàn)證初篩陽(yáng)性信號(hào)分子的蛋白表達(dá)情況。結(jié)果1.Ad DLX1和Ad RFP分別感染MG63細(xì)胞后,與RFP對(duì)照組相比,Ad DLX1能顯著增加骨肉瘤細(xì)胞MG63中DLX1的表達(dá)。2.MG63細(xì)胞中DLX1過(guò)表達(dá)后,與RFP對(duì)照組相比,細(xì)胞遷移能力和侵襲能力下降。3.MG63細(xì)胞中DLX1過(guò)表達(dá)后,與RFP對(duì)照組相比,過(guò)表達(dá)DLX1對(duì)細(xì)胞增殖、凋亡無(wú)明顯影響。4.MG63細(xì)胞中DLX1過(guò)表達(dá)后,與RFP對(duì)照組相比,過(guò)表達(dá)DLX1對(duì)細(xì)胞成骨分化無(wú)明顯影響。5.MG63細(xì)胞中DLX1過(guò)表達(dá)后,與RFP對(duì)照組相比,DLX1過(guò)表達(dá)可上調(diào)β-catenin m RNA和蛋白的表達(dá)。結(jié)論1.MG63細(xì)胞中DLX1過(guò)表達(dá)后,對(duì)MG63細(xì)胞的惡性生物學(xué)行為有不同程度的影響:可抑制MG63細(xì)胞的遷移和侵襲,對(duì)MG63細(xì)胞的增殖和凋亡無(wú)顯著影響;對(duì)MG63細(xì)胞成骨分化無(wú)明顯促進(jìn)作用。2.DLX1在MG63細(xì)胞中過(guò)表達(dá)后,Wnt/β-catenin信號(hào)通路參與DLX1對(duì)MG63細(xì)胞生物學(xué)行為的調(diào)控。第二部分DLX1聯(lián)合BMP9對(duì)人骨肉瘤細(xì)胞惡性生物學(xué)行為及成骨分化的影響目的探討DLX1聯(lián)合BMP9對(duì)人骨肉瘤細(xì)胞MG63惡性生物學(xué)行為及成骨分化的影響。方法采用構(gòu)建有DLX1和BMP9基因的重組腺病毒Ad DLX1和Ad BMP9,單獨(dú)或聯(lián)合感染人骨肉瘤MG63細(xì)胞,分為RFP組(Ad RFP感染組)、BMP9組(Ad BMP9+Ad RFP感染組)、DLX1+BMP9組(Ad DLX1+Ad BMP9感染組)、DLX1組(Ad DLX1+Ad GFP感染組),觀察細(xì)胞熒光表達(dá)情況并以RT-PCR和Western blot驗(yàn)證DLX1、BMP9m RNA和蛋白表達(dá)情況;Transwell實(shí)驗(yàn)檢測(cè)MG63細(xì)胞遷移和侵襲;DAPI染色和流式細(xì)胞術(shù)檢測(cè)MG63細(xì)胞凋亡;CCK-8檢測(cè)MG63細(xì)胞增殖;ALP染色和ALP讀數(shù)分析細(xì)胞早期成骨能力,Western blot檢測(cè)骨橋蛋白(osteopontin,OPN)的蛋白表達(dá),茜素紅染色檢測(cè)細(xì)胞晚期成骨能力;采用RT-PCR初篩相關(guān)信號(hào)通路,Western blot驗(yàn)證初篩陽(yáng)性信號(hào)分子的蛋白表達(dá)情況。結(jié)果1.Ad DLX1單獨(dú)感染MG63細(xì)胞后,細(xì)胞中DLX1的m RNA和蛋白表達(dá)上調(diào);Ad BMP9單獨(dú)感染MG63細(xì)胞后,細(xì)胞中BMP9的m RNA和蛋白表達(dá)上調(diào);Ad DLX1和Ad BMP9聯(lián)合感染MG63細(xì)胞后,細(xì)胞中DLX1、BMP9的m RNA和蛋白表達(dá)均上調(diào)。2.與RFP組相比,BMP9組、DLX1+BMP9組、DLX1組中MG63細(xì)胞遷移和侵襲能力均下降,且DLX1+BMP9組對(duì)遷移和侵襲能力的抑制較BMP9組和DLX1組有所增加。3.與RFP組相比,BMP9組、DLX1+BMP9組中MG63細(xì)胞增殖能力均下降,且兩組MG63細(xì)胞受到的增殖抑制無(wú)明顯差異;DLX1組細(xì)胞增殖較RFP組無(wú)明顯差異。4.與RFP組相比,BMP9組、DLX1+BMP9組、DLX1組中MG63細(xì)胞凋亡無(wú)明顯差異。5.與RFP組相比,BMP9組中MG63細(xì)胞ALP活性、OPN蛋白表達(dá)和鈣鹽沉積有所增加,但增加程度有限;DLX1+BMP9組中MG63細(xì)胞ALP活性增強(qiáng)、OPN蛋白表達(dá)上調(diào)、鈣鹽沉積明顯增加;DLX1組中MG63細(xì)胞ALP活性、OPN蛋白表達(dá)和鈣鹽沉積無(wú)明顯增加。結(jié)論1.DLX1單獨(dú)作用于人骨肉瘤細(xì)胞MG63時(shí),可抑制MG63細(xì)胞遷移和侵襲,對(duì)MG63細(xì)胞凋亡和增殖無(wú)明顯影響;不能促進(jìn)MG63細(xì)胞成骨分化。2.BMP9單獨(dú)作用于人骨肉瘤細(xì)胞MG63時(shí),可抑制MG63細(xì)胞遷移、侵襲和增殖,對(duì)MG63細(xì)胞凋亡無(wú)明顯影響;對(duì)MG63細(xì)胞成骨分化的促進(jìn)作用較弱。3.DLX1與BMP9聯(lián)合作用后,可抑制MG63細(xì)胞遷移、侵襲和增殖,對(duì)MG63細(xì)胞凋亡無(wú)明顯影響;可促進(jìn)MG63細(xì)胞成骨分化。4.骨肉瘤是一種分化異常導(dǎo)致的惡性增生物,DLX1聯(lián)合BMP9作用后可降低人骨肉瘤細(xì)胞遷移、侵襲和增殖的惡性生物學(xué)行為,促進(jìn)人骨肉瘤細(xì)胞成骨分化。
[Abstract]:The effect of overexpression of DLX1 on the malignant biological behavior and osteogenic differentiation of human osteosarcoma cells in order to explore the effect of overexpression of DLX1 on the malignant biological behavior and osteogenic differentiation of human osteosarcoma cell MG63. Methods the recombinant adenovirus (adenovirus, Ad) Ad DLX1 and Ad RFP of the empty adenovirus were used to infect people respectively. Osteosarcoma cell MG63 was divided into DLX1 experimental group (Ad DLX1 infection group) and RFP control group (Ad RFP infection group). The expression of cell fluorescence was observed and the expression of DLX1 was tested with RT-PCR and Western blot. Alkaline phosphatase (alkaline phosphatase, ALP) staining and ALP reading were used to analyze the early osteogenesis of MG63 cells. Western blot was used to detect the protein expression of osteopontin (osteopontin, OPN). Alizarin red staining was used to detect the late osteogenesis of the cells; RT-PCR initial screening related signal pathways, Western blot to verify the protein expression of the positive signal molecules of the initial screening. Results after 1.Ad DLX1 and Ad RFP infected MG63 cells respectively, compared with the RFP control group, Ad DLX1 significantly increased the expression of DLX1 in the MG63 cells of osteosarcoma cells after the DLX1 over expression, and compared with the control group, the cell migration ability and invasion ability decreased. After cell proliferation, apoptosis had no obvious effect on the overexpression of DLX1 in.4.MG63 cells. Compared with the RFP control group, overexpression of DLX1 had no significant effect on DLX1 over expression in.5.MG63 cells. Compared with the RFP control group, the over expression of DLX1 could up regulate the beta -catenin m RNA and the expression of protein. The malignant biological behavior has different effects: it can inhibit the migration and invasion of MG63 cells, have no significant effect on the proliferation and apoptosis of MG63 cells; there is no obvious promoting effect on the osteogenic differentiation of MG63 cells. After the overexpression of.2.DLX1 in MG63 cells, Wnt/ beta -catenin signaling pathway and DLX1 regulate the biological behavior of MG63 cells by DLX1. Second parts. The effect of DLX1 combined with BMP9 on the malignant biological behavior and osteogenic differentiation of human osteosarcoma cells in order to explore the effect of DLX1 combined with BMP9 on the malignant biological behavior and osteogenic differentiation of human osteosarcoma cells MG63. Methods the recombinant adenovirus Ad DLX1 and Ad BMP9, which had DLX1 and BMP9 genes, were used to infect human osteosarcoma MG63 cells alone or in combination. They were divided into group RFP (Ad RFP infection group), group BMP9 (Ad BMP9+Ad RFP infection group), DLX1+BMP9 group (Ad DLX1+Ad BMP9 infection group), DLX1 group (Ad DLX1+Ad). MG63 cell apoptosis was detected by cytometer, MG63 cell proliferation was detected by CCK-8, ALP staining and ALP reading were used to analyze the early osteogenic ability of cells, Western blot was used to detect the protein expression of osteopontin (osteopontin, OPN), and alizarin red staining was used to detect the late osteogenesis of the cells; RT-PCR initial screening signal pathway was used, and Western blot was used to verify the positive signal of initial screening. Results when 1.Ad DLX1 infecting MG63 cells alone, the expression of M RNA and protein expression of DLX1 in the cells was up-regulated, and Ad BMP9 was infected with MG63 cells alone, and m RNA and protein expression of BMP9 in the cells were up regulated. The migration and invasion ability of MG63 cells in group BMP9, group DLX1+BMP9 and group DLX1 decreased, and the inhibition of migration and invasion in group DLX1+BMP9 was higher than that in group BMP9 and DLX1 group, and the proliferation ability of MG63 cells in BMP9 group and DLX1+BMP9 group decreased, and there was no significant difference in proliferation inhibition between the two groups. There was no significant difference between group RFP and group RFP. Compared with group RFP, there was no significant difference in the apoptosis of MG63 cells in group BMP9, DLX1+BMP9 and DLX1. The MG63 cell ALP activity, the increase of protein expression and calcium salt deposition in the BMP9 group increased, but the increase degree was limited. The ALP activity of MG63 cells, OPN protein expression and calcium salt deposition in DLX1 group did not increase obviously. Conclusion 1.DLX1 can inhibit the migration and invasion of MG63 cells in human osteosarcoma cells alone, and have no obvious effect on the apoptosis and proliferation of MG63 cells, and can not promote the MG63 cell osteogenic differentiation.2.BMP9 alone to act on the human osteosarcoma cell MG63. It could inhibit the migration, invasion and proliferation of MG63 cells, and had no obvious effect on the apoptosis of MG63 cells. The combination of.3.DLX1 and BMP9 could inhibit the migration, invasion and proliferation of MG63 cells, and had no obvious effect on the apoptosis of MG63 cells, and could promote the differentiation of.4. osteosarcoma of MG63 cells to be a kind of abnormal differentiation. DLX1 combined with BMP9 can reduce the malignant biological behavior of human osteosarcoma cell migration, invasion and proliferation, and promote osteosarcoma cells osteogenic differentiation.

【學(xué)位授予單位】：重慶醫(yī)科大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2017
【分類號(hào)】：R738

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